Blood and Marrow Draw Processing Devices and Methods

ABSTRACT

Apparatus, systems and methods for processing a blood sample. One embodiment includes an isolation container having at least one sidewall defining an interior volume. The interior volume includes a medial reservoir in fluid communication with proximal and distal reservoirs. The diameter of the medial reservoir is less than the diameter of the proximal and distal reservoirs. Therefore, the isolation container has an interior volume which is roughly hour-glass shaped with the medial reservoir being a substantially narrowed portion or channel between two wider portions. The isolation container is configured such that the buffy coat layer of fractionated blood will be located within, or may be manipulated to be with the medial reservoir.

This application is a continuation of U.S. application Ser. No.14/767,679 filed on Aug. 13, 2015, entitled “Blood and Marrow DrawProcessing Devices and Methods”, which is a 35 U.S.C. § 371 nationalphase application of PCT/US14/16814 (WO 2014/130426), filed on Feb. 18,2014, entitled “Blood and Marrow Draw Processing Devices and Methods”,which application claims the benefit of U.S. Provisional ApplicationSer. No. 61/767,385, filed Feb. 21, 2013, which is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to devices and methods for drawing bloodor marrow from a patient, separating the blood into density gradedfractional layers and isolating selected layers. The disclosedembodiments more particularly may be used to draw, separate and isolatethe buffy coat layers from a peripheral blood, marrow blood or wholemarrow sample.

BACKGROUND OF THE INVENTION

There are many procedures and processes that require the separation ofblood into blood components or density graded layers. As used herein,“blood” may include peripheral blood, marrow blood or whole marrow.Known methods include density gradient additives which expand portionsof the blood, discrete sample centrifugation, and continuous centrifugalseparation. Separation of blood products may be required for diagnostictests, blood donation, transfusions, or autogenic therapeutic reasons.The constituent components of a fractioned blood sample, listed fromupper-most (least dense) to lower-most (highest density) fractions whena density separation is viewed from the side, are as follows:platelet-poor plasma, platelet-rich plasma, the “buffy coat” and redblood cells (RBCs). The buffy coat is the separated blood portion thatcontains the white blood cells, platelets, mesenchymal stem cells,hematopoietic stem cells, macrophages, adipocytes, osteoblasts,endothelial progenitor cells, very small embryonic like stem cells,blastomere like stem cells, and other nucleated cells.

Several techniques have been developed to isolate of the above notedfractions. Some of these techniques are substantially automated andperformed by machine. Other techniques use substantially manual andfeature centrifugation followed by some degree of manual post-processingto isolate the fractions. Alternative techniques involve manualisolation of centrifuged blood sample fractions using standardlaboratory equipment and aseptic techniques. For example, as shown inFIG. 1 (Prior Art) a 50 cc or other appropriately sized conical tube isoften used with centrifugation to fractionate a blood sample. There arebenefits and shortcomings to each of the conventional bloodfractionation methods. Some of the metrics used as indicators of theeffectiveness of an isolation performance are as follows; time requiredto process blood, percentage of available fraction isolated, technicianskill required to successfully produce quality isolations, andcross-contamination risk between isolated fractions.

As noted above, one blood fraction of interest is the buffy coat. Thebuffy coat is the separated portion of an un-coagulated blood samplethat contains most of the nucleated cells, including but not limited towhite blood cells, platelets, mesenchymal stem cells, hematopoietic stemcells, macrophages, adipocytes, osteoblasts, endothelial progenitorcells, very small embryonic like stem cells, blastomere like stem cells,and other nucleated cells after density gradient centrifugation.Typically, the buffy coat makes up less than 1% of the total volume of ablood sample. Although the buffy coat is predominately composed of whiteblood cells and platelets, the buffy coat also contains the varioustypes of stem cells listed above. Stem cells, including mesenchymal stemcells (MSCs) are pluripotent blast or embryonic-like cells located inblood, bone marrow, dermis and perisosteum. In general these cells arecapable of renewing themselves over extended periods of time as well as,under various environmental conditions, differentiating into cartilage,bone and other connective tissue. In this manner MSCs and other types ofstem cells have been reported to have regenerative capabilities in anumber of animal models.

Further, these finding are being extended in clinical trials to humans.Typical MSC therapies must be initiated with a source of autologous ornon-autologous MSCs, and the proposed therapy can feature in vitro or invivo MSC expansion. In view of the relatively tiny volume of the buffycoat and the relatively tiny quantity of MSCs in the buffy coat, it isuseful to efficiently extract as much of the buffy coat from a sample asis possible in an aseptic and waste free manner

The present invention is directed toward overcoming one or more of theproblems discussed above.

SUMMARY OF THE EMBODIMENTS

The embodiments disclosed herein include apparatuses, systems andmethods for processing a blood sample. One apparatus embodimentcomprises an isolation container having at least one sidewall definingan interior volume. The interior volume of the isolation containerincludes a proximal reservoir having a select diameter. The interiorvolume also includes a medial reservoir in fluid communication with theproximal reservoir. The diameter of the medial reservoir is less thanthe diameter of the proximal reservoir. The interior volume alsoincludes a distal reservoir in fluid communication with the medialreservoir with the distal reservoir having a diameter which is greaterthan the diameter of the medial reservoir. Therefore the isolationcontainer has an interior volume which is roughly hour-glass shaped withthe medial reservoir being a substantially narrowed portion or channelbetween two wider portions.

The isolation container also includes a plunger in sealing and slidableengagement with the side walls of the distal reservoir and a coupling influid communication with the proximal end of the proximal reservoir. Theplunger may in certain instances be a one-piece element fabricated fromplastic or another material, more typically however; the plunger mayinclude a sealing element fabricated from an elastomeric material inslidable engagement with the side walls and a handle attached to thesealing element. In use, a portion of the handle will initially extendbeyond the distal end of the distal reservoir. Thus, the plunger anddistal reservoir may be manipulated in the manner of a conventionalsyringe to draw a fluid such as blood into the interior volume of theisolation container.

In certain embodiments, at least a portion of the handle may beselectively removed from the remaining portion or portions of theplunger. For example, the handle may include a series of perforationsdefining one or more break-lines at which a portion of the handle may beconveniently removed. A break-line or break-plane formed as describedabove may also be used to define a specific selected volume within theisolation container. For example, the break-plane may be made coplanarwith a plane defined by the distal end of the sidewall of the distalreservoir, thus defining a selected volume within the isolationcontainer.

Alternative embodiments of the apparatus for processing a blood sampleinclude an isolation container having a port which opens into the medialreservoir. Alternatively, the isolation container may include aninternal or external lumen which opens into the medial reservoir. Incertain embodiments the position of the port or lumen opening may beadjusted axially toward or away from the proximal and distal reservoirs.

In use, blood, bone marrow or combination of blood and marrow may bedrawn into the isolation container by withdrawing the plunger. The bloodor marrow may be drawn from any mammalian blood or marrow source. Theisolation container may then be sealed or capped and placed directlyinto a centrifuge to fractionate the blood or marrow into density gradedlayers. The isolation container is sized such that the buffy coat layerof fractionated blood will be located within the reduced cross sectionalmedial reservoir after the centrifuge step. In this manner the axiallength of the buffy coat is increased, facilitating efficientwithdrawal. It is known that the percentage of hematocrits (red bloodcells) in a blood sample will vary widely from patient to patient. Thus,a blood processing system may include a selection of two, three orseveral isolation containers having various internal volumes andinternal volume configurations which may be matched with sample size andpatient hematocrit levels to assure proper placement of the buffy coatafter centrifugation. Alternatively, or in conjunction with theselection of an appropriately sized isolation container, the samplevolume may be adjusted to place the layer of interest, for example thebuffy coat, within the reduced diameter medial reservoir where the buffycoat is most accessible after centrifugation is complete.

After the centrifuge step, a technician may withdraw the buffy coat fromthe balance of the sample through the port or lumen if a port or lumenis provided, or using a pipette, needle, suction or other techniquewhich can be used to access the medial reservoir.

The disclosed embodiments are particularly useful for drawing marrow orblood from a patient, processing the sample on site to isolate the buffycoat and re-injecting the buffy coat and mesenchymal stem cells (MSCs)contained therein into the patient for therapeutic purposes. Thus, thedisclosed embodiments are optimized to minimize sample transfers, samplewaste, contamination risk and processing delays.

In certain apparatus embodiments it will be useful for a technician tovisually observe the location of the buffy coat within the medialreservoir. Accordingly, certain apparatus embodiments are provided withat least a medial reservoir sidewall manufactured from a transparentmaterial.

Alternative apparatus embodiments include but are not limited tostand-alone isolation containers substantially as described above butwithout a proximal coupling. In this embodiment blood or marrow may beplaced into the isolation container for further processing, directlyfrom a conventional syringe. Alternatively, an isolation containerhaving a proximal coupling and a distal coupling may be placed in-linebetween a trocar, needle, catheter or other fluid pathway and aconventional syringe such that the isolation container may be filled bycausing a partial vacuum in the entire system by operating the syringe.

Several system embodiments are also disclosed. System embodimentsinclude an isolation container or series of volume graded isolationcontainers plus a trocar, needle, catheter, associated tubing and otherelements required to place the isolation container into fluidcommunication with a source of blood or marrow. The system may alsoinclude a centrifuge configured to directly receive the isolationcontainer eliminating the need to transfer the blood or marrow sample toa separate centrifuge container.

Alternative disclosed embodiments include methods of using the disclosedsystems and apparatus to process a blood sample. In particular, methodembodiments include drawing or otherwise placing blood or marrow into anisolation container, centrifuge and the contents of the isolationcontainer and withdrawing a selected density-graded layer (typically thebuffy coat) from the isolation container. The method may further includeinjecting the buffy coat, or a portion of the buffy coat, for exampleMSCs, into a patient for therapeutic purposes. The disclosed apparatusthus provides a method for withdrawing, processing and effectivelywithdrawing a portion of a fractionated blood or marrow sample using oneisolation container for several processing steps, thus minimizing wasteand contamination risk. In addition, the steps of the disclosed methods,including re-injecting material into a patient for therapeutic purposesmay be performed in one visit to a single treatment facility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of blood fractioned in a prior artprocessing tube.

FIG. 2 is a schematic illustration of a system for processing blood.

FIG. 3 is a plan cross sectional view of a isolation container asdisclosed.

FIG. 4 is a plan sectional view of a plunger as disclosed.

FIGS. 5A, 5B, and 5C are a series of plan view illustrations of a bloodprocessing apparatus as disclosed in various operational states.

FIGS. 6A and 6B are plan cross sectional views of an alternativeisolation container embodiment featuring a port or magnifying elementassociated with a sliding collar.

FIG. 7 is a plan cross sectional view of an alternative isolationcontainer embodiment featuring a lumen.

FIG. 8 is a schematic illustration of blood fractioned in isolationcontainer as disclosed.

FIG. 9 is schematic illustration of an alternative blood processingsystem as disclosed.

FIG. 10 is schematic illustration of an alternative blood processingsystem as disclosed.

FIG. 11 is a graph comparing the results of a marrow draw and separationperformed with conventional techniques compared to a marrow draw andseparation performed with selected disclosed techniques.

FIG. 12 is a graph showing the improvement realized with a marrow drawand separation performed with conventional techniques compared to amarrow draw and separation performed with selected disclosed techniques.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments disclosed herein include an apparatus for processing a bloodor marrow sample, various blood sample processing systems, and methodsof processing a blood sample. The disclosed apparatus, systems andmethods may be used to draw blood from a mammalian patient, separate theblood into density graded layers and isolate layers of interest. Thedisclosed apparatus, systems and methods are optimized to minimize thenecessity for transferring a blood sample from or between variouscontainers during processing. A “blood sample” is defined herein as aquantity of blood drawn from a mammal including but not limited to ahuman. The blood sample may be a peripheral blood sample drawn from avein or artery. Alternatively the blood sample may be a marrow bloodsample or whole marrow drawn from a source of marrow within bone tissue.

FIG. 2 schematically illustrates an apparatus for processing blood 100in a system configured for use. As shown in FIG. 2, the apparatus 100may be connected to a source of marrow, blood or both, for example, theiliac crest 102 of a patient's pelvis. The apparatus may be connected tothe source of blood using a trocar 104, needle, catheter or othermethod. Similarly, the apparatus 100 may be connected to a source ofperipheral blood using known techniques.

As more particularly shown in FIGS. 3-4, the apparatus 100 may includean isolation container 106 and a plunger 108. The isolation container106 includes at least one sidewall 110 defining an interior volume. Theinterior volume of the isolation container 106 includes at least threeseparate regions in fluid communication with each other. As shown inFIG. 3, the interior volume includes a proximal reservoir 112 having aselect diameter d. The interior volume also includes a medial reservoir114 in fluid communication with the proximal reservoir 112. The medialreservoir 114 has a diameter d′ which is less than the select diameter dof the proximal reservoir 112. The interior volume also includes adistal reservoir 116 in fluid communication with the medial reservoir114 opposite the proximal reservoir 112. The distal reservoir 116 has adiameter d″ which is greater than the diameter d′ of the medialreservoir 114. Although the diameters d and d″ are illustrated as beingroughly equivalent in FIG. 3, this configuration is not required. Asdescribed in detail below the respective diameters of the variousreservoirs which comprise the isolation container can be selected toachieve desired blood processing results. In all cases however, thediameter d′ of the medial reservoir 114 will be substantially less thanthe diameters of the remaining reservoirs. Furthermore, the termdiameter as used herein does not require that the various reservoirshave a circular cross-section. Alternative embodiments may haveelliptical or irregular cross-section defining at least one diameter.

The apparatus 100 also includes a plunger 108. One embodiment of plunger108 includes a sealing element 118 and a handle 120 which may be made ofdistinct materials joined together. For example, the sealing element 118may be made of rubber, silicone or another compliant material and thehandle may be made of a plastic of selected rigidity. Plungerembodiments consisting of only one material or more than two materialsare within the scope of this disclosure.

In use, the plunger 108 is received in sealing and slidable engagementwith the side wall 110 of the distal reservoir 116. Thus, at least theperiphery of the sealing element 118 and potentially more of the plunger108 forms a substantially fluid-tight seal with the inner wall of distalreservoir 116. A seal is made while maintaining the ability of theplunger 108 to slide lengthwise toward or away from the medial andproximal reservoirs. Thus, the plunger 108 and distal reservoir 116 inthe FIG. 3-4 embodiment function like a syringe such that moving theplunger 108 away from the medial and proximal reservoirs causes apartial vacuum which can be employed to draw a blood sample into theisolation container 106.

The FIG. 3-4 embodiment also includes a coupling 122 in fluidcommunication with the proximal reservoir 112. The coupling may be, butis not limited to, a standard Luer-type coupling which facilitatesattachment to a trocar, catheter, needle or other device. At certaintimes during the processing of a blood sample, the coupling 122 may besealed with a cap 124.

FIG. 4 provides a detailed view of one embodiment of plunger 108. Asnoted above the plunger may include a compliant sealing element 118 andhandle 120. The handle may include a grip structure 126 which, in use,extends beyond a distal sidewall surface 128 of the distal reservoir116. It may be advantageous for effective blood sample processing toremove a portion of the handle 120 (or a portion of plunger 108 insingle plunger material embodiments) prior to performing certainprocessing steps. For example, the isolation container 106 and plunger108 may fit more readily into a centrifuge if a portion of the handle120 is removed. Alternatively, a system may include a centrifugeconfigured to accept the isolation container 106 and plunger 108 withouthandle removal or modification.

Thus, in some embodiments, the handle 120 or plunger 108 may be providedwith a plurality of perforations 130, a score line, a thinned region, aglue line, a region of relatively weak material, socket and coupling,male and female screw threading or other structure which defines abreak-line at which a portion of the handle may be selectively removedfrom a remaining portion of the handle. In the FIG. 4 embodiment, theperforations 130 continue in the handle structure extending out from theprinted page, thus the perforations 130 define multiple break lineswhich together define a break-plane. Alternatively, the plunger mayinclude a handle which can be unscrewed from the sealing element orotherwise disengaged from the sealing element or a lower handle portion.

As illustrated in FIGS. 5A, 5B, and 5C, the break plane (or anothersurface defined when a portion of the handle is removed, for example byun-screwing the handle) may be used to set and control a specific samplesize volume within the isolation container 106. For example, as shown inFIG. 5A, the sealing member 118 of the plunger 108 may be placed firmlyagainst the proximal edge of the distal reservoir 116 prior to drawingblood into the isolation container 106. Then, as shown in FIG. 5B, theplunger 108 may be withdrawn until the perforations or other elementsdefining a break-line are beyond the distal edge 128 of the distalreservoir 116. As then shown in FIG. 5C, The outer portion of the handlemay be removed from the balance of the plunger. At this point, thebreak-plane defined by the perforations 130 may be made co-planar withthe distal edge of the distal reservoir to precisely control the volumewithin the isolation container 106, between the proximal end of thecoupling 122 and the proximal surface of the plunger 108.

FIG. 6A illustrates an alternative embodiment of isolation container 106further comprising a port 132 passing through a portion of the isolationcontainer wall 110 defining the medial reservoir 114. Thus, said port132 defines an opening directly into the medial reservoir 114 fromoutside the isolation container 106 without passing through the proximalreservoir 112 or the distal reservoir 116. As detailed below, the medialreservoir can serve to isolate the buffy coat or other blood fraction ofinterest. In embodiments including a port 132, the port facilitates theaccurate withdrawal or removal of the buffy coat from other layers. Incertain embodiments, the port 132 includes a collar 134, slot, lumen orother mechanism which allows the location of the port 132 to betranslated axially toward the proximal reservoir or axially toward thedistal reservoir to facilitate removal of the buffy coat from otherlayers.

FIG. 6B illustrates an alternative embodiment where a magnifying lens135 is associated with the isolation container wall 110 at the medialreservoir 114. In certain embodiments, the magnifying lens 135 may beslid longitudinally along the medial reservoir 114 to aid a technicianwith the task of identifying and subsequently effectively removing therelatively small buffy coat layer from the sample. A magnifying lens 135may be a simple cylinder-type magnifier fabricated from glass ortransparent plastic in conjunction with a sliding collar 134 as shown inFIG. 6B. Alternatively the magnifying lens could be a more complexoptical element having precision lens components. In certainembodiments, the magnifying lens 135 and a port 132 may both be providedas a single structure or multiple structures operatively connected to asingle collar 134.

FIG. 7 illustrates an embodiment of isolation container 106 including aninner lumen 136 having an opening at one end which is in fluidcommunication with the medial reservoir 114. In selected embodimentsfeaturing an inner lumen 136, the position of the inner lumen openingmay be translated within the medial reservoir axially toward or awayfrom the proximal and distal reservoirs. The inner lumen 136 may be usedto efficiently remove the buffy coat or other selected blood fractionfrom other layers.

Certain embodiments of isolation container 106 will be subjected to oneor more manual or operator guided processing steps. Accordingly, it maybe advantageous in certain embodiments to fabricate the sidewall 110, inat least the region of the medial reservoir 114, from an opticallytransparent material.

The isolation container 106 may (in conjunction with a plunger 108) andother apparatus such as a trocar, be used to directly drawn marrow orperipheral blood from a patient. Alternatively, as noted below, otherembodiments of isolation container may receive blood drawn by othermeans. In either case, the isolation container 106 may be part of ablood sample processing system. As shown in FIG. 2, a system may includethe apparatus 100 and in particular an isolation container 106, a trocar104 or other fluid pathway to a source of blood, and a centrifuge 138.The centrifuge 138 may directly receive the isolation container 106 tominimize blood handling, contamination risk and transfer waste. Prior toplacement in the centrifuge 138 the openings to the isolation container106 may be sealed with a cap 124 and plunger sealing elements 118 asdescribed above or through other means. The blood contained within theisolation container 106 may then be centrifuged such that the sampledevelops density-graded layers within the isolation container.

As shown in FIG. 8, the isolation container 106 will typically be sizedsuch that the buffy coat will be located within the reduced crosssectional medial reservoir 114 independent of hematocrits after acentrifuge step. In this manner the axial size of the buffy coat isincreased, facilitating withdrawal. It is known that the percentage ofhematocrits (red blood cells) in a blood sample will vary widely frompatient to patient. Thus, a blood processing system may include aselection of two, three or several isolation containers 106 havingvarious internal volumes and configurations which may be matched withsample size and patient hematocrit levels. Alternatively, or inconjunction with the selection of an appropriately sized isolationcontainer 106, the sample volume may be adjusted to place the layer ofinterest, for example the buffy coat, within the reduced diameter medialreservoir 114 where the buffy coat is most accessible aftercentrifugation is complete. The medial reservoir 114 of the isolationcontainer will typically occupy 10-20% of the total volume of thecontainer. The remaining volume will be split between the proximalreservoir and distal reservoir of the isolation container with theproximal reservoir containing 1-8% more volume than the distalreservoir, because RBC percentage of blood ranges from 42-48%.

The disclosed embodiments are particularly useful for drawing marrow orblood from a patient, processing the sample on site and re-injecting thebuffy coat and MSCs into the patient for therapeutic purposes. Thus, thedisclosed embodiments are optimized to minimize sample transfers, waste,contamination risk and processing delays.

After the isolation container 106 has been centrifuged for apredetermined amount of time, it may be removed from the centrifuge. Atechnician may remove the cap 124 or seal and inserts a probe, needle,pipette or other device into the medial reservoir 114 to draw the buffycoat from the sample. In embodiments featuring a port 132 or inner lumen136, the buffy coat or other layer of interest may be directly withdrawnthrough the port or inner lumen. If a magnifying element 133 is providedas shown in FIG. 6B, the technician may use the magnifier to bettervisualize the buffy coat and assure complete and efficient removal ofsame. One skilled in the art will recognize that there are a variety ofmethods and devices that may be used to draw the fluid buffy coat fromthe isolation container 106. A standard syringe may be used to drawfluid out of the isolation container, a serological pipetter may be usedto draw fluid into a pipette, a powered vacuum system operated by atechnician may be used to draw fluid into a reservoir, or a systememploying siphon functionality may be used to draw fluid into areservoir.

FIG. 9 illustrates an alternative embodiment where blood is drawn into astandard syringe 902 using standard methods. The syringe 902 may then bedetached from the trocar and the blood contained in the syringe isemptied into an isolation container 904 which may not have a proximalcoupling. The isolation container functions as described above and iscentrifuged such that the buffy coat or other layer of interest islocated in the reduced diameter medial reservoir 906 of the device. Thetechnician can now use one of the many techniques described herein toremove the buffy coat from the blood sample.

FIG. 10 shows another system embodiment. An intermediate isolationcontainer 1002 is operatively positioned between a standard syringe 1004and a trocar 1006, needle, catheter or other conduit inserted into thepatient. The syringe 1004 is operated such that blood is drawn into theintermediate isolation container 1002. Once a predetermined amount ofblood has been drawn into the isolation container, the syringe 1004 isdisconnected from the isolation container 1002. A cap or suitable sealis placed over the distal opening 1008 of the isolation container. Inaddition, the isolation container 1002 is disconnected from the trocar1006. A cap or suitable seal is placed over the proximal connector 1010.In certain embodiments, a flexible tube may be employed such that theisolation container 1002 is able to be oriented such that the proximalconnector opening is vertical, preventing spillage of the fluid when thedisconnection is made. The container 1002 may then be placed into acentrifuge and spun as described above. A technician may use proceduresdescribed herein to remove the buffy coat.

The devices, apparatus, systems and methods described herein may be usedto process a blood sample such that the buffy coat may be efficientlyisolated and extracted. Mesenchymal stem cells (MSCs) are predominantlylocated within the buffy coat of a blood or whole marrow sample. MSCsare pluripotent blast or embryonic-like cells located in blood, bonemarrow, dermis and periosteum. In general these cells are capable ofrenewing themselves over extended periods of time as well as, undervarious environmental conditions, differentiating into cartilage, boneand other connective tissue.

EXAMPLES Example 1 Comparison of Nucleated Cell (Buffy Coat) Recoveryusing Conventional and Disclosed Techniques

FIGS. 11-12 graphically illustrate the enhanced effectiveness ofcollecting a buffy coat when using the methods, apparatus and systemsdescribed herein as compared to standard techniques used to collect thebuffy coat from a conventionally processed blood or marrow sample.

Conventional Technique:

A marrow draw sample is typically sent to a processing laboratory fromthe procedure room. The marrow sample of Example 1 comprises two 30 ccsyringes for a total marrow volume of 60 cc to be processed. There wasalso extracted an additional 10 cc syringe of marrow that was used forquality assurance, but not used in the sample preparation. The marrowwas placed into two (2) 50 cc conical tubes similar to those illustratedin FIG. 1. These conical tubes were placed into a standard labcentrifuge and centrifuged at a force of 200 g (calculated from rotordiameter and RPM) for 6 minutes. A BD Falcon serological pipette wasused to remove the buffy coat from the two 30 mL conical tubes andsubsequently the buffy coat was placed into a 15 mL conical tube. This15 mL conical tube was placed onto the centrifuge and spun for 6 minutesat 200 g. After this second centrifuge step, there was about 1 mL ofbuffy coat material within the 15 mL conical tube. The operator drew outthe buffy coat with a serological pipetter resulting in about 1.5 mL ofmaterial for analysis.

Disclosed Technique

The disclosed technique was tested with a prototype isolation containerand plunger similar to that disclosed in FIGS. 1-3. The testing of thedevice did not involve drawing marrow from a live patient, although theprototype was designed with that capability. As noted above, thedisclosed embodiments are particularly useful for drawing marrow orblood from a patient, processing the sample on site and re-injecting thebuffy coat and MSCs into the patient for therapeutic purposes. Thus, thedisclosed embodiments are optimized to minimize sample transfer, wasteand processing delays.

Since the device was not tested on a live patient, marrow was insertedinto the device through the distal opening. The volume of marrowinserted into the device was accurately measured so that positioning ofthe buffy coat would fall within the reduced diameter medial reservoirof the isolation container after use of the centrifuge. The prototypeincluded a template for trimming stock plungers in a manner similar tothat shown in FIGS. 5A, 5B, and 5C so that exactly 30 mL of marrow wouldbe processed by the system. After the marrow was inserted into theisolation container, it was spun in the centrifuge for 15 minutes at 740RPM (125 g). Thus, the force applied to the sample was slightly slowerand longer than the standard technique because of issues with theplunger moving out of position due to centrifugal force. After theseparation, the isolation container was removed from the centrifuge. Theoperator used a syringe and needle to contemporaneously draw off thebuffy coat. Both 5 and 10 mL syringes and the needles were used for theexamples.

Results

Four samples were processed according to the two techniques describedabove. As shown in FIG. 11, the number of nucleated cells extracted fromeach sample with the disclosed technique exceeded the number ofnucleated cells extracted using conventional techniques. As shown inFIG. 12, the disclosed techniques resulted in a 1.7 fold to 4.6 foldincrease in nucleated cell recovery. See also Table 1 below.

TABLE 1 Nucleated Cells/30 cc Sample Conventional Technique DisclosedTechnique Sample 1 197.5 e⁶ NCs/30 cc 339.0 e⁶ NCs/30 cc Sample 2  56.7e⁶ NCs/30 cc 109.5 e⁶ NCs/30 cc Sample 3 102.0 e⁶ NCs/30 cc 476.0 e⁶NCs/30 cc Sample 4 130.2 e⁶ NCs/30 cc 360.0 e⁶ NCs/30 cc

What is claimed is:
 1. An isolation container for processing a bloodsample comprising: a proximal reservoir having a diameter; a transparentmedial reservoir in fluid communication with the proximal reservoir, thetransparent medial reservoir having a diameter which is less than thediameter of the proximal reservoir; a distal reservoir positionedopposite the transparent medial reservoir from the proximal reservoir,the distal reservoir being in fluid communication with the transparentmedial reservoir, the distal reservoir having a diameter which isgreater than the diameter of the transparent medial reservoir; and anadjustable sealing element in sealing engagement with a portion of aside wall of the distal reservoir, said adjustable sealing elementproviding for an increase or a decrease of a volume within the distalreservoir.
 2. The isolation container for processing a blood sample ofclaim 1 further comprising a handle engaged with the sealing element. 3.The isolation container for processing a blood sample of claim 2 furthercomprising a threaded connection between the handle and the sealingelement.
 4. The isolation container for processing a blood sample ofclaim 1 further comprising a removable cap closing an opening into theproximal reservoir.
 5. The isolation container for processing a bloodsample of claim 1 further comprising a magnifying lens operativelyassociated with the transparent medial reservoir.
 6. A blood sampleprocessing system comprising: an isolation container comprising: aproximal reservoir having a diameter; a transparent medial reservoir influid communication with the proximal reservoir, the transparent medialreservoir having a diameter which is less than the diameter of theproximal reservoir; a distal reservoir positioned opposite thetransparent medial reservoir from the proximal reservoir, the distalreservoir being in fluid communication with the transparent medialreservoir, the distal reservoir having a diameter which is greater thanthe diameter of the transparent medial reservoir; and an adjustablesealing element in sealing engagement with a portion of a side wall ofthe distal reservoir, said adjustable sealing element providing for anincrease or a decrease of a volume within the distal reservoir; and asample withdrawal apparatus configured to withdraw a fluid from theisolation container.
 7. The blood sample processing system of claim 6further comprising a handle engaged with the sealing element.
 8. Theblood sample processing system of claim 7 further comprising a threadedconnection between the handle and the sealing element.
 9. The bloodsample processing system of claim 8 further comprising a removable capclosing an opening into the proximal reservoir.
 10. The blood sampleprocessing system of claim 6 further comprising a magnifying lensoperatively associated with the transparent medial reservoir.
 11. Theblood sample processing system of claim 6 further comprising acentrifuge comprising sample opening sized to receive the isolationcontainer.
 12. The blood sample processing system of claim 6 wherein thesample withdrawal apparatus comprises a syringe and a hollow needle. 13.A method of processing a blood sample comprising: placing the bloodsample into an isolation container comprising; a proximal reservoirhaving a diameter; a transparent medial reservoir in fluid communicationwith the proximal reservoir, the transparent medial reservoir having adiameter which is less than the diameter of the proximal reservoir; adistal reservoir positioned opposite the transparent medial reservoirfrom the proximal reservoir, the distal reservoir being in fluidcommunication with the transparent medial reservoir, the distalreservoir having a diameter which is greater than the diameter of thetransparent medial reservoir; and an adjustable sealing element insealing engagement with a portion of a side wall of the distalreservoir, said adjustable sealing element providing for an increase ora decrease of a volume within the distal reservoir; engaging theisolation container with a centrifuge; centrifuging the blood samplewithin the isolation container; and withdrawing a selected bloodconstituent from the transparent medial reservoir with a samplewithdrawal apparatus.
 14. The method of claim 13 wherein the selectedblood constituent is a buffy coat.
 15. The method of claim 13 furthercomprising causing the selected blood constituent to flow from thedistal reservoir to the transparent medial reservoir by adjusting theposition of the sealing element to cause a decrease in the volume withinthe distal reservoir.
 16. The method of claim 13 further comprisingcausing the selected blood constituent to flow from the proximalreservoir to the transparent medial reservoir by adjusting the positionof the sealing element to cause an increase in the volume within thedistal reservoir.
 17. The method of claim 13 further comprising removinga handle engaged with the sealing element by unthreading a threadedconnection between the handle and the sealing element.
 18. The method ofclaim 13 further comprising closing an opening into the proximalreservoir with a removable cap prior to centrifuging the blood withinthe isolation container.
 19. The method of claim 18 further comprising:providing a sample withdraw apparatus comprising a syringe and a hollowneedle; removing the cap from the opening into the proximal reservoir;viewing the selected blood constituent through a transparent side wallof the transparent medial reservoir; inserting the hollow needle throughthe opening into the proximal reservoir and into fluid communicationwith the selected blood constituent in the transparent medial reservoir;and withdrawing the selected blood constituent into the hollow needlewith the syringe.
 20. The method of claim 19 further comprising viewingthe selected blood constituent through a magnifying lens operativelyassociated with the transparent side wall of the transparent medialreservoir.